RESUMEN
Functional assays have been used to define the estrogenicity of xenoestrogens in cotransfection studies employing estrogen receptors in various cell lines. It is known that estrogen is able to affect transcription from other nuclear transcription factors, especially the nuclear factor-kappa B (NF-kappa B). The ability of selected xenoestrogens (methoxychlor [MXC], dieldrin, and o',p'-DDT) to transrepress the NF-kappa B-mediated transcription in Cos-1 cells was evaluated by cotransfection of human estrogen receptor-alpha (hERalpha). These xenoestrogens have been described as comparably potent xenoestrogens, whereas their relative binding activity (RBA) has been relegated to a lower order as compare to estrogen. The two NF-kappa B response element-containing SV40 promoter and -242/+54 cytomegalovirus (CMV)-expressing firefly luciferase (2 x NRE-PV-Luc and 2 x NRE-CMV-Luc, respectively) were transfected into Cos-1 cells with pRL-tk, expressing the renilla luciferase as internal control. The estrogen receptor was expressed from cytomegalovirus major immediate early promoter (CMV-MIEP) (CMV5-hERalpha). Treatment with 1 nM estrogen (E(2)) (26.2%), 5 nM E(2) (41.4%; p < .05), and xenoestrogens (methoxychlor [1 nM: 29.6%, p < .05; 10 nM: 22.6%), dieldrin [1 nM: 10.3%; 10 nM: 36.06%, p < .05], and o',p'-DDT [1 nM: 17.0%; 10 nM: 7.15%]) repressed transcription from 2 x NREX-PV-Luc. The antiestrogen, ICI 182,780, failed to antagonize the effects of xenoestrogens. The effects of xenoestrogens in transrepression of NF-kappa B by ERalpha were similar when 2 x NRE-CMV-Luc was employed as reporter. Statistically significant (p < .01) repression by 1 nM E(2) (69.2%), 5 nM E(2) (69.1%), 1 nM o',p'-DDT (51.4%), 1 nM dieldrin (47.3%), and 1 nM MXC (73.3%) were observed. The effect of these xenoestrogens without ERalpha cotransfection on 2 x NRE-PV-Luc- and 2 x NRE-CMV-Luc-mediated NF-kappa B transcription was not affected by the treatment alone. It is concluded that xenoestrogens, like estrogens, are capable of producing transrepression of NF-kappa B by hERalpha.
Asunto(s)
Receptor alfa de Estrógeno/fisiología , FN-kappa B/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Animales , Células COS , Chlorocebus aethiops , Dieldrín/farmacología , Ensayo de Cambio de Movilidad Electroforética , Estradiol/análogos & derivados , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/genética , Estrógenos/farmacología , Fulvestrant , Regulación de la Expresión Génica/efectos de los fármacos , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Metoxicloro/farmacología , FN-kappa B/genética , Ésteres del Forbol/farmacología , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Elementos de Respuesta/genética , Acetato de Tetradecanoilforbol/farmacología , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
Plasmids of the pT181 family encode initiator proteins that act as dimers during plasmid rolling circle (RC) replication. These initiator proteins bind to the origin of replication through a sequence-specific interaction and generate a nick at the origin that acts as the primer for RC replication. Previous studies have demonstrated that the initiator proteins contain separate DNA binding and nicking-closing domains, both of which are required for plasmid replication. The tyrosine residue at position 191 of the initiator RepC protein of pT181 is known to be involved in nicking at the origin. We have generated heterodimers of RepC that consist of different combinations of wild type, DNA binding, and nicking mutant monomers to identify the role of each of the two monomers in RC replication. One monomer with DNA binding activity was sufficient for the targeting of the initiator to the origin, and the presence of Tyr-191 in one monomer was sufficient for the initiation of replication. On the other hand, a dimer consisting of one monomer defective in DNA binding and the other defective in origin nicking failed to initiate replication. Our results demonstrate that the monomer that promotes sequence-specific binding to the origin must also nick the DNA to initiate replication. Interestingly, whereas Tyr-191 of the initiator was required for nicking at the origin to initiate replication, it was dispensable for termination, suggesting that alternate amino acids in the initiator may promote termination but not initiation.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Replicación del ADN , Plásmidos/genética , Dimerización , Conformación ProteicaRESUMEN
A large number of plasmids have been shown to replicate by a rolling circle (RC) mechanism. The initiators encoded by these plasmids have origin-specific, nicking-closing activity that is required for the initiation and termination of RC replication. Since the initiators of many RC plasmids are rate-limiting for replication, these proteins are usually inactivated after supporting one round of replication. In the case of the pT181 plasmid, inactivation of the initiator RepC protein occurs by the attachment of an oligonucleotide to its active tyrosine residue. We have generated the inactivated form of RepC, termed RepC*, in vitro and investigated the effects of attachment of the oligonucleotide on its various biochemical activities. Our results demonstrate that while RepC* is inactive in nicking-closing and replication activities due to the blockage of its active tyrosine residue, it is competent in origin DNA binding and DNA religation activities. We have investigated the oligomeric state of RepC and RepC* and found that RepC exists as a dimer in solution and can oligomerize on the DNA. We have generated heterodimers in vitro between the wild-type and epitope-tagged RepC proteins. In electrophoretic mobility shift experiments, the initiator heterodimers generated a novel DNA-protein complex, demonstrating that it binds to DNA as a dimer. We have shown that a DNA binding mutant of RepC can be targeted to the origin in the presence of the wild-type protein primarily through a protein-protein interaction. Interestingly, RepC* is defective in its ability to oligomerize on the DNA. RepC* inhibited the DNA binding and replication activity of wild-type RepC to only a very limited extent, suggesting that it may play only a minor regulatory role in replication in vivo. Based on these and earlier results, we propose a model for the role of RepC during the initiation and termination of pT181 RC replication.
Asunto(s)
Proteínas Bacterianas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Oligonucleótidos/farmacología , Origen de Réplica/efectos de los fármacos , Secuencia de Bases , Centrifugación por Gradiente de Densidad , ADN Bacteriano/metabolismo , Dimerización , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos/metabolismo , Tirosina/metabolismoRESUMEN
The role of renal nonprotein sulfhydryls (NPSH) in mercuric chloride-induced nephrotoxicity has been studied in various laboratories. Similarly, the importance of NPSH for mercuric ion accumulation by renal tissue also has been studied. In this study the potential role of NPSH was examined with respect to mercuric ion effects on membrane transport utilizing isolated membrane vesicles prepared from Sprague-Dawley rat kidneys. Sodium gradient-driven p-aminohippurate (PAH) transport in basolateral vesicles and glucose transport in brush border vesicles were studied. Depletion of NPSH, primarily glutathione (GSH), appeared to alter PAH but not glucose transport. HgCl2 (1 mg/kg) had no effect on either transport system in vesicles isolated from kidneys with normal GSH content, but it markedly disrupted both PAH and glucose transport in vesicles isolated from GSH-depleted rats. The most consistent effects were observed after GSH depletion with diethyl maleate plus buthionine sulfoximine. Elevation of renal GSH by administration of glutathione monoethyl ester blocked the effect of mercuric chloride (4 mg/kg) on glucose transport reported earlier. These data indicate that renal sulfhydryls not only modulate the effects of mercuric chloride, but they also may be important for normal physiological functioning of the PAH transport system.
Asunto(s)
Membrana Celular/fisiología , Glutatión/metabolismo , Riñón/efectos de los fármacos , Cloruro de Mercurio/farmacología , Animales , Membrana Basal/efectos de los fármacos , Membrana Basal/metabolismo , Transporte Biológico/efectos de los fármacos , Butionina Sulfoximina , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Sinergismo Farmacológico , Glucosa/metabolismo , Glutatión/análogos & derivados , Glutatión/farmacología , Glutatión/fisiología , Riñón/metabolismo , Riñón/fisiología , Masculino , Maleatos/farmacología , Cloruro de Mercurio/efectos adversos , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacología , Ratas , Ratas Endogámicas , Ácido p-Aminohipúrico/metabolismoRESUMEN
The hepatoprotective activity of picroliv, the irridoid glycoside mixture from Picrorhiza kurrooa, was determined in adult male albino rats. Pretreatment with picroliv prevented the hepatotoxic effects of paracetamol and galactosamine as evidenced by various biochemical and histopathological observations. Maximum hepatoprotective effect was observed with daily oral doses of 6 and 12 mg/kg for 7 or 8 days. The antihepatotoxic action of picroliv seems likely due to an alteration in the biotransformation of the toxic substances resulting in decreased formation of reactive metabolites.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Cinamatos/farmacología , Glicósidos/farmacología , Plantas Medicinales/química , Ácido Vanílico/farmacología , Acetaminofén/toxicidad , Animales , Galactosamina/toxicidad , India , Hígado/efectos de los fármacos , Pruebas de Función Hepática , Masculino , Extractos Vegetales/farmacología , Ratas , Ratas Endogámicas , Silimarina/toxicidadRESUMEN
Both chromate and citrinin have been shown to produce acute renal damage. Although both substrates act on the proximal tubule in the rat, they affect different parts of that nephron segment. As with most nephrotoxicants, the mechanism(s) or subcellular target(s) for citrinin or chromate is unknown. The availability of methodology for isolation of functional membrane vesicles has afforded the opportunity to study the plasma membrane as a target for the effects of citrinin and chromate. Whether studied solely with in vitro conditions or after administration to the rat, chromate exhibited its primary action on the basolateral (BL) membrane vesicles. This was exhibited by a reduction in the p-aminohippurate (PAH) overshoot. At both 3 and 16 hr after treatment (40 mg/kg, sc) there was a significant, but relatively modest, effect on glucose transport by brush border (BB) vesicles. Citrinin, when studied in vitro, inhibited PAH transport (BL vesicles), but had only equivocal effects on BB glucose transport. However, after pretreatment of the rats with citrinin (60 mg/kg, ip), both BL and BB membrane vesicle function was reduced markedly at 3 hr. By 16 hr, an overshoot had returned for both transport substrates, although the glucose overshoot was still significantly below control. These data demonstrate that both citrinin and chromate alter proximal tubular cell membrane function and do so relatively early after administration to the rat. This effect suggests that alteration of membrane function by these nephrotoxicants is an early, if not initiating, event in the production of acute tubular necrosis.
Asunto(s)
Cromatos/toxicidad , Citrinina/toxicidad , Riñón/efectos de los fármacos , Compuestos de Potasio , Animales , Transporte Biológico Activo/efectos de los fármacos , Glucemia/metabolismo , Técnicas In Vitro , Riñón/metabolismo , Masculino , Membranas/efectos de los fármacos , Membranas/metabolismo , Microvellosidades/efectos de los fármacos , Ratas , Ratas Endogámicas , Ácido p-Aminohipúrico/metabolismoRESUMEN
Both brush border and basolateral membrane vesicles were prepared from rat kidney by Percoll gradient centrifugation. The addition of mercuric chloride (100 nM) to vesicles prepared from healthy, male, Sprague-Dawley rats reduced p-aminohippurate (PAH) transport by basolateral vesicles. No effect was observed on glucose transport by brush border vesicles even at mercuric chloride concentrations as high as 10 microM. However, when the metal salt was added in the presence of 5% bovine serum albumin, basolateral PAH transport was unaffected. Transport studies also were done with vesicles isolated from rats pretreated with mercuric chloride (4 mg/kg, sc). Transport of PAH was unaffected at all times studied. Glucose transport was unaffected at 1 and 3 hr, but at 16 hr was reduced significantly. By 48 hr, brush border glucose transport had recovered. These data demonstrate that mercuric chloride can alter renal membrane function, and that the effects depend on the membrane vesicle population used. With pretreatment studies, the time after treatment also influences whether or not an effect is seen.
Asunto(s)
Riñón/efectos de los fármacos , Cloruro de Mercurio/toxicidad , Animales , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Técnicas In Vitro , Riñón/metabolismo , Masculino , Cloruro de Mercurio/metabolismo , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismo , Unión Proteica , Ratas , Ratas Endogámicas , Albúmina Sérica Bovina/farmacología , Ácido p-Aminohipúrico/farmacocinéticaAsunto(s)
Riñón/efectos de los fármacos , Metales/toxicidad , Compuestos de Sodio , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/fisiología , Cromatos/toxicidad , Glucosa/farmacocinética , Riñón/metabolismo , Riñón/ultraestructura , Proteínas de la Membrana/metabolismo , Cloruro de Mercurio/toxicidad , Microvellosidades/metabolismo , Microvellosidades/ultraestructura , Proteínas de Transporte de Monosacáridos/metabolismo , Ratas , Ácido p-Aminohipúrico/farmacocinéticaRESUMEN
The protective effects of atropine, diacetylmonoxime (DAM), and diazepam separately and in combination were investigated in rats exposed to malathion. Malathion (500 mg/kg, ip) inhibited acetylcholinesterase (AchE) activity in RBC and brain and produced hyperglycemia and hyperlactacidemia with depletion of glycogen in liver, triceps, and brain of animals 2 hr after its administration. Atropine (20 mg/kg, ip) given immediately after malathion abolished hyperglycemia and glycogenolytic effect but exhibited no effect on the recovery of inhibited AchE activity. DAM (100 mg/kg ip) given immediately after malathion significantly reactivated the inhibited AchE activity both in RBC and brain. It also partially modified hyperglycemia and glycogenolytic effect. Diazepam (50 mg/kg, ip) slightly modified AchE and abolished hyperglycemia, hyperlactacidemia, and glycogenolytic effects. A combination of these drugs protected the animals from the acute toxic effects of malathion.
Asunto(s)
Atropina/uso terapéutico , Butanonas/uso terapéutico , Reactivadores de la Colinesterasa/uso terapéutico , Diacetil/uso terapéutico , Diazepam/uso terapéutico , Malatión/envenenamiento , Acidosis Láctica/inducido químicamente , Acidosis Láctica/tratamiento farmacológico , Animales , Encéfalo/enzimología , Inhibidores de la Colinesterasa/envenenamiento , Diacetil/análogos & derivados , Quimioterapia Combinada , Eritrocitos/enzimología , Glucógeno/metabolismo , Hiperglucemia/inducido químicamente , Hiperglucemia/tratamiento farmacológico , Glucógeno Hepático/metabolismo , Malatión/antagonistas & inhibidores , Masculino , RatasRESUMEN
Diazinon, an organophosphorous compound, produced hyperglycemia and reduced the glycogen content of the brain 2 h after its administration to rats (40 mg/kg, i.p.). The activities of the glycogenolytic enzymes, glycogen phosphorylase and phosphoglucomutase, were significantly increased, while that of glucose-6-phosphatase was not altered. Atropine (20 mg/kg, i.p.) given immediately after diazinon abolished the changes; tolazoline or propranolol (each at 10 mg/kg, i.p.) injected 30 min before the administration of diazinon significantly reduced the hyperglycemia and the increase in brain glycogenolysis. A combination of tolazoline and propranolol was more effective than either of them alone and completely abolished the hyperglycemia and the changes in brain glycogenolysis. It may be concluded that diazinon initially activates central cholinergic processes leading to hyperglycemia and increased cerebral glycogenolysis in animals.
Asunto(s)
Encéfalo/metabolismo , Diazinón/farmacología , Glucógeno/metabolismo , Hiperglucemia/metabolismo , Insecticidas/farmacología , Animales , Atropina/farmacología , Encéfalo/efectos de los fármacos , Femenino , Glucosa-6-Fosfatasa/metabolismo , Fosfoglucomutasa/metabolismo , Fosforilasas/metabolismo , Propranolol/farmacología , Ratas , Ratas Endogámicas , Tolazolina/farmacologíaAsunto(s)
Cinamatos/farmacología , Glicósidos/farmacología , Hidroxibenzoatos/farmacología , Hepatopatías/tratamiento farmacológico , Ácido Vanílico/farmacología , Animales , Cinamatos/aislamiento & purificación , Femenino , Galactosamina , Glicósidos/aislamiento & purificación , Hepatopatías/etiología , Malaria/complicaciones , Masculino , Muridae , Plasmodium berghei , Ratas , Ratas Endogámicas , Ácido Vanílico/aislamiento & purificaciónRESUMEN
Pseudallescheria boydii infection involving the left distal femur and knee joint developed in a patient with Cushing's disease, and recurred after treatment with both intravenous miconazole and intravenous amphotericin B. The patient subsequently had synovectomy and arthrodesis, and received ketoconazole, 600 mg per day orally for one year. Concurrent with the start of ketoconazole, a bilateral adrenalectomy was done to control endogenous hypercortisolism. The patient's infection then resolved, and one year afterward there is no clinical or radiologic evidence of recurrent infection.
Asunto(s)
Artritis Infecciosa/complicaciones , Síndrome de Cushing/complicaciones , Micetoma/complicaciones , Osteomielitis/complicaciones , Adrenalectomía , Artritis Infecciosa/terapia , Terapia Combinada , Humanos , Cetoconazol/uso terapéutico , Articulación de la Rodilla/microbiología , Masculino , Miconazol/uso terapéutico , Persona de Mediana Edad , Micetoma/diagnóstico , Micetoma/terapia , Osteomielitis/terapia , Pseudallescheria/aislamiento & purificación , RecurrenciaRESUMEN
Endosulfan was administered orally (2.5 and 7.5 mg/kg) daily to male rats for a period of 60 days. The distribution pattern of alpha and beta-isomers was studied using a gas-liquid chromatograph equipped with an electron capture detector. There was a significant increase in liver and lung weights. The testes weight was slightly decreased. No significant change in weights of other tissues was observed. At both dose levels, the concentration of alpha-isomer was highest in kidney (574 and 1655 ng/g, respectively), followed by lung, ventral prostate, spleen, testes and brain. In the seminal vesicle, epididymis, heart and liver, the concentration of beta-isomer was higher than the alpha-isomer. The results of the study indicated a differential ability to accumulate the two isomers of endosulfan which may help to explain the difference in the toxic potential of the alpha- and beta-isomers.